Lysosomal sulphate transport is dependent upon sulphydryl groups.
Using thiol blocking agents, we examined the role of sulphydryl groups for function of the lysosomal sulphate transport system. Monothiol binding reagents, p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulphonic acid (p-CMBS), dithiol binding reagents such as CuCl2, the alkylating agent, N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in Km to 867 microM versus a control value of 126 microM and a modest decrease in Vmax to 99 pmolperunit beta-hexosaminidase per 30 s versus a control value of 129 pmolperunit beta-hexosaminidase per 30 s. Similar although somewhat less dramatic results were obtained using Cu2+ with an increase of Km to 448 microM and a Vmax of 77 pmolperunit beta-hexosaminidase per 30 s. The sulphate transport activity of detergent solubilized lysosomal membranes could be bound to a p-chloromercuribenzoic acid (p-CMB)-Sepharose sulphydryl affinity resin and eluted with mercaptoethanol. Sulphydryl groups thus appear to play a role in sulphate transport through effects on substrate affinity. Sulphydryl-binding appears to be a strategy that may be useful for purification of the transporter.
Chou
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HF
Division of Medical Genetics, E4, Department of Pediatrics, Harbor-UCLA Medical Center, 1124 W. Carson St., Torrance, CA 90502, USA.
Passage
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Jonas
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AJ
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003300:Copper / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013431:Sulfates / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013431:Sulfates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D003300:Copper; D005033:Ethylmaleimide; D004229:Dithiothreitol